Tumor, particularly malignant tumor, is a disease which cause serious harm to human health in today's world, and is the 2nd deadly among all diseases. But in recent years, the incidence rate was significantly increased. The malignant cancer has poor treatment, accompanied with high metastasis rate at late stage and poor prognosis. Current conventional clinical treatment methods including radiotherapy, chemotherapy and surgery, which although largely alleviate the pain and prolong the survival time, have significant limitations, and are difficult to improve their efficacy further.
Proliferation of normal cells is strictly controlled by respective ligands activating their growth factor receptors, such as growth factor receptor tyrosine kinases. Cancer cell proliferation is also through its factor receptor activation, but it loses the strict control of normal proliferation. This loss of control may be caused by many reasons, such as growth factor over-expression, overexpression of growth factor receptors, or spontaneous activation of biochemical pathways regulated by growth factors. Oncogenic receptors include epidermal growth factor receptor (EGFR), platelet derived growth factor receptor (PDGFR), insulin-like growth factor receptor ((IGFR), nerve growth factor receptor (NGFR), and fibroblast growth factor receptor ((FGF) etc.
Epidermal growth factor receptor (EGFR) is also known as c-erbB1/HER1, whose family members are growth factor receptor tyrosine kinases, their cell surface with specific growth factors or natural ligand interactions, such as with EGF or TGF ci interactions, thereby activating the receptor tyrosine kinases. The first member of the family has been found to be a glycoprotein with apparent molecular weight of 165 KD.
EGFR plays an important role in the regulation of tumor cell growth, repair and survival, angiogenesis, invasion and metastasis, and is expressed in a considerable number of human tumors. In many malignant tumors, the expression of EGFR is often associated with a poor prognosis and a low survival rate. Based on this, if there is a drug which can block EGFR activity, it will inhibit the phosphorylation and signal transduction, thus play an anti-tumor functions in multiple aspects, and increase the anti-tumor chemotherapy and radiotherapy treatment. In some studies, EGFR inhibitors show addictive and synergistic effects when used in combined treatment with various chemotherapy drugs and radiation therapy drugs for certain cancers.
EGFR inhibitors include monoclonal antibodies, tyrosine kinase inhibitors, quinazoline pyrrolo-/pyrrolo-/pyridopyridines, ligand-toxin and immunotoxin complexes, as well as antisense oligonucleotides and EGFR/ligand mediated vaccines.
It was demonstrated in some in vivo and in vitro experiments that the anti-EGFR antibody can successfully inhibit the growth of EGFR-expressing tumor cell lines. In treatment of solid tumors, the results from some anti-EGFR monoclonal antibodies alone or their combination with traditional treatment methods are encouraging.
Glycosylation is a protein important post-translational modifications. Protein molecular surface sugar chains can have a profound impact on the structure and function of the protein molecules, glycosylation as an important post-translation process, has a great impact on proper proteins folding, localization, immunogenicity and biological activity. The glycosylation and glycan structure of mAb antibody have strong correlation with its function, by affecting the binding of IgG molecules to FcRs, Clq and FeRn to regulate the antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) and half-life of IgG molecules. Glycosylation also affects the safety features of mAb, particularly non-human glycans, and has potential immunogenicity. The glycans located in Fab functional region can affect both the safety and efficacy features of these drugs.
Glycosylation is highly dependent on cell expression system and subclone selection, and many factors during cell culture, for example medium components, culture conditions will affect glycosylation, thereby affecting the biological activity, efficacy, immunogenicity and pharmacokinetics of therapeutic proteins.
Among the therapeutic monoclonal antibodies currently marketed, the vast majority is produced by recombinant DNA technology, and the vast majority use in vitro cell culture technology. Because of the complexity of mammalian cell structure, function and gene expression regulation, there is a big difference between the expression of exogenous genes in mammalian cells and that in prokaryotes, consequently, the machinery for efficient expression of exogenous genes is also different from that for prokaryotes cells. Expression of exogenous gene in mammalian cells includes gene transcription, mRNA translation and post-translational modifications etc. Post-translational modifications include glycosylation, phosphorylation, oligomerization, as well as the formation of intra- or intermolecular disulfide bonds between protein molecules. Post-translational modification is crucial to the function of the protein, so it may be necessary to express certain proteins with biological functions in mammalian cells, such as membrane proteins, antibodies and enzymes having specific catalytic function. CHO cells and mouse myeloma cells (NS0, SP2/0) expression system has currently become the golden standard as cell engineering system for therapeutic antibody and Fc-fusion proteins. According to statistics, 48% of currently approved therapeutic monoclonal antibodies are expressed in CHO cells, while 45% are expressed in murine cells (21% NS0 cells, 14% SP2/0 cells, 10% hybridoma cells). Although the integrity of polypeptide chains in different expression systems and culture conditions seems unchanged, the changes of glycosylation types cannot be ignored.
Cetuximab (Erbitux®, C225 mab), is a recombinant chimeric monoclonal antibody specifically targeting epidermal growth factor receptor (EGFR), and was approved in many countries for the treatment of metastatic colorectal cancer and head and neck squamous cell carcinoma. However, a number of studies have reported that the drug hypersensitivity reactions occur at very high incidence in clinical applications. Drug specific IgE antibodies were found in the serum of most patients with hypersensitivity reactions, and it specifically reacts against α-Gal. Further research found that, Erbitux® is expressed and prepared in mammalian cells (mouse myeloma cells SP2/0), and this murine cell line containing an additional α1,3-galactosidase transferase, which primarily mediates the transfer of galactose residue is from UDP-Gal of a conformation to the terminal galactose residues, thereby generating α-Gal. α-Gal is a harmful non-human disaccharide, found in certain glycans on mAb, especially mAb expressed in the murine cell lines. High levels of anti-α-Gal IgE antibodies were found in some patients. If using mAb with glycan containing α-Gal units for treatment, there will be serious hypersensitivity reactions. Further, the difference of murine cell IgG glycosylation from human is that, murine cells not only have the biosynthetic machinery to produce α-Gal epitope, but also produce N-hydroxyethyl neuraminidase (NGNA), rather than N-acetyl phenol neuraminidase (NANA). The distinction of NGNA and NANA is there is an additional oxygen atom in NGNA, and glycoproteins are considered to be closely associated with the immunogenicity in humans if they contain NGNA residues. Some marketed therapeutic glycoproteins have cause serious adverse reactions in the patients because they contains NGNA residues.